Chapter 5 MR haplotypes modulate stress responses Materials and methods

Functional characterization in vitro

Construction of the hMR plasmids

The expression plasmid containing human MR was obtained from Dr. R. Evans (gene expression laboratory and HHMI, The Salk Institute for Biological Studies, La Jolla, Ca) and is described elsewhere (Arriza et al., 1987).

MR-2G/C (rs2070951) and MRI180V (rs5522) sites were mutated from G to C and from A to G, respectively using primers 5’-GGCCGAGGCAGCGATGGAGACCAAAG-3’ and

5’-CGCTGCCTCGGCCCTTTGGTCTCCAT-3’ and primers 5’-GGCGTCATGCGCGCCGTTGTTAAAAGCCCCTAT-3’ and

5’-ATAGGGCTTTTAACAACGGCGCGCATGACGCC-3’ and the Quick Change Site Directed Mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s protocol.

After mutagenesis the hMR insert of the plasmid was sequenced to assure absence of other mutations.

Transactivation assay

Cos-1 cells (African green monkey kidney cells) were cultured in DMEM high glucose supplemented with 10% FCS (Gibco, Paisley, UK). Cells were seeded in 24-well plates (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) at 3 x 104 cells/well in DMEM supplemented with charcoal-stripped serum.The cells were transfected the next day using SuperFect (Qiagen, Venlo, The Netherlands). hMR plasmids and the reporter pasmid TAT3-Luc (tyrosine amino transferase triple hormone response element) were used at 100 ng/well. The control plasmid pCMV-R (Promega, Leiden, The Netherlands) coding forRenilla luciferase controlled by cytomegalovirus (CMV) promoter was used (10 ng/well). One day after transfection, the cells were treated with cortisol (Sigma-Aldrich, Zwijndrecht, the Netherlands) in concentrations ranging from 0 to 10-8 _M. After 24h of incubation the cells were harvested in passive lyses buffer (Promega) and firefly and Renilla luciferase activity was determined using a dual label reporter assay (Promega) and a luminometer (CENTRO XS3 LB960, Berthold, Bad Wildbad, Germany). Three separate experiments were performed and all three experiments were performed in triplicate.

Western blot

For western blot Cos-1 cells were seeded in6-well plates (Greiner Bio-One, Alphen a/d Rijn, The Netherlands)at 2x105 cells/well in DMEM supplemented with charcoal-stripped serum.The cells were transfected the next day using Trans-it Cos transfection reagent (Mirus, Madison, USA). Plasmids containing one of the hMR variants or no hMR (control) were used at 2µg/well. Cells were harvested 48 hours after transfection. The primary antibody MR 1D5 (a generous gift by Gomez-Sanchez, Division of endocrinology, University of Mississippi, Jackson, MS) was diluted 1:1000 in 0.5 % milk powder in Tris buffered saline and Tween 20 (TBST) and incubated for 1h at room temperature (RT). The secondary antibody goat anti-mouse IgG HRP was used in 1:5000 dilutions in TBST with 0.5 % milk for 1 h at RT. Tubulin was used as a control for the amount of cells and the monoclonal anti -Tubulin was used at a 1:1000 dilution (T6557; Sigma-Aldrich, Zwijndrecht, the Netherlands). The ECL detection system (GE healthcare, Diegem, Belgium) was used for detection. The differences in intensity of the MR bands were quantified with Image J

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Chapter 5 MR haplotypes modulate stress responses

(ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/). Three separate experiments were performed.

Ligand binding assay

Cos-1 cells were seeded in20 cm plates (Greiner Bio-One, Alphen a/d Rijn, The Netherlands)at 2x106 cells/plate in DMEM supplemented with 5% charcoal-stripped serum.Cells were transfected the next day using Mirus TransIt- COS reagent according to the manufacturer’s protocol (Sopachem, Ochten, The Netherlands) and hMR plasmids were usedat 30 µg/plate. After 24 hours medium was replaced with serum free DMEM and after another 24 hours cells were pelleted. All further steps are carried out at 0°C. Cells were resuspended in 3.5ml buffer (5mM Tris-HCl (pH 7.4), 1mM EDTA, 1mM B-Mercaptoethanol, 10mM Na-Molybdate, 5% glycerol) per plate and 3 x 15 seconds homogenised using an electric homogenizer (Pro200, Pro scientific, Oxford, CT, USA). The homogenate was centrifuged (100.000 x g, 2°C) to obtain cytosol.

200µl cytosol was incubated with [3H]Cortisol (70 Ci/mmol, Amersham, Buckinghamshire, UK) to asses total binding or [3H]Cortisol and a 500 fold excess of dexamethasone (Sigma-Aldrich, Zwijndrecht, the Netherlands) to asses non-specific binding. [3H]Cortisol was used at 0.5nM, 1nM, 1.5nM, 2.5nM, 3.5nM, 5nM. After vortexing and 3 hours incubation on ice bound and free [3H]Cortisol fractions were separated by Sephadex LH-20 as described previously (de Kloet et al., 1975). Fractions containing the receptor bound radioligand were collected, vortexed with 3ml Ultima Gold scintillation fluid (Perkin Elmer, Waltham, Massachusetts, USA) and radioactivity was measured in a liquid scintillation analyzer (1900CA Packard, Perkin Elmer). Three separate experiments were performed and all three experiments were performed in triplicate.

Statistical analysis

The in vitro experiments were analyzed using GraphPad prism 4 (GraphPad software Inc, San Diego, CA). In the transactivation assays firefly/renilla luciferase ratios were normalized against the highest signal and background expression was subtracted. MR protein expression measured by western blot was normalized against -Tubulin. The differences between the four hMR variants were analyzed with one and two-way ANOVAs with Bonferroni posttests. In the radioligand binding assay one-binding-site curve fitting was used to determine the dissociation constant (Kd) and maximal binding (Bmax). The specific MR cortisol binding was obtained by subtracting the non-specific binding from the total binding. The difference in Kd and Bmax between MRI180 and MR180V was tested with a t-test. In vitro results are shown as the mean  SD.

Genetic association study

Recruitment

We approached teachers of all major school types in the region of Trier (Germany) and Luxembourg by means of personal visits in local schools and by newspaper announcements. Teachers were entered into the study if they reported to be free of psychiatric disorders, diabetes, pregnancy, and corticosteroid or psychotropic medication. Written informed consent was obtained from all participants and the protocol was approved by the ethics committee of the University of Trier and the Rheinland-Pfalz State Medical Association.